Project Summary The PI?s LONG-TERM GOAL is to develop a cure for Transthyretin Amyloidosis (ATTR), an age-related disease associated with heart failure, neuropathy, and ultimately death. ATTR is a protein misfolding disorder in which monomeric Transthyretin protein (TTR) misfolds, assembles into amyloid, and infiltrates the heart and peripheral nerves. Internalization and cytoplasmic accumulation of TTR aggregates leads to an intracellular stress response by the proteostasis system, such as activation of the chaperone and autophagy systems. Since proteostasis become less efficient with age, a lower intracellular response may contribute to age- associated cytotoxicity. Current treatments focus on limiting the amount of monomeric TTR protein, yet do not address the impact of pre-existing amyloid aggregates, including changes in proteostasis. Therefore, there is an urgent need to identify new approaches to disassemble intracellular aggregates and prevent the re-seeding of monomeric TTR protein. The OBJECTIVE OF THIS PROJECT is to investigate how the proteostasis system can be used to enhance TTR aggregate disassembly and clearance, while limiting the re-seeding of monomeric TTR. Our RATIONALE is that uncovering ways to disassemble TTR aggregates and prevent their re-seeding will provide novel directions for developing effective therapies to treat ATTR patients. The slow onset of TTR aggregation makes study in traditional higher eukaryote models both time consuming and expensive. Here, we have established a yeast model system that provides a rapid and economic means to address three SPECIFIC AIMS: 1) Determine how the proteostasis machinery impacts intracellular TTR aggregate disassembly and clearance, including a novel baiting strategy to help target in vivo disaggregation of TTR amyloids. 2) Identify dominant negative mutations that prevent further intracellular TTR aggregation, by using the yTRAP system to assess potential capping of TTR aggregates by mutant TTR alleles, thereby preventing further conversion of monomeric to aggregated TTR. 3) Determine TTR sequence requirements for heterologous cross-seeding, by testing which sequences within the TTR protein foster the cross-seeding of TTR aggregates from other amyloids. The PROPOSED RESEARCH IS INNOVATIVE because A) our novel approach overcomes current limitations in studying intracellular TTR aggregation in higher eukaryotes, and B) can be used to develop approaches that disassemble and/or degrade intracellular TTR amyloid and limit the re-seeding of TTR. The PROPOSED PROJECT IS SIGNIFICANT because the identification of effective factors that dissolve TTR amyloid will provide a strong framework for impactful therapeutic directions.